This proposal is concerned with further studies of in vitro culture systems able to support the various developmental forms of salivarian trypanosomes (Trypanosoma brucei sspp. and T. congolense) found in the vertebrate and invertebrate hosts. First, attention will be given to the reproduction of the entire life cycle of trypanosomes in culture. Bloodstream forms from established cultures of T. b. brucei and T. b. rhodesiense, incubated at 37 C, will be used to initiate cultures of the vector forms (procyclic, epimastigote, and metacyclic) incubated at 26-28 C. In vitro-produced metacyclic forms will serve as starting material for the cultivation of the life cycle of T. congolense. In the second part of the proposal the emphasis will be on the cultivation of metacyclic forms currently (in the case of T. brucei sspp.) available only from tsetse fly tissue cultures. Studies will include: (a) the possible substitution of tissues of nonhematophagus Diptera for those of tsetse fly in the T. brucei sspp. metacyclic-form culture system; (b) attempts at cultivation of metacyclic T. brucei sspp. in association with various Glossina and mammalian cell lines cultivated at 26-28 C; and (c) the design of a culture medium based on the chemical analyses of tsetse fly saliva for the growth of the forms found in the salivary glands (T. brucei) and proboscis (T. congolense). Comparisons between the various stages of T. brucei and T. congolense life cycles produced in vitro with those developing in vivo will be studied by light and electron microscopy. Furthermore, the MVATs of the metacyclic stages of T. brucei produced in vitro and in the tsetse flies infected with the same stocks will be compared by immunological tests. There is a need for additional methods to cultivate infective forms of salivarian trypanosomes, which may ultimately be used in developing new approaches to immuno- and chemotherapy.